Gametes are specialized haploid cells (e.g., spermatozoa and oocytes) produced by meiosis and involved in sexual reproduction. By contrast, diploid cell has its chromosomes in homologous pairs, and has two copies of each autosomal genetic locus. The diploid number (2n) equals twice the haploid number and is the characteristic number for most cells other than gametes. A zygote is the diploid cell resulting from the fusion of male and female gametes during fertilization. THE DICTIONARY OF CELL BIOLOGY 103, 139, 388 (J. M. Lackie et al., eds. 1995). Only a (diploid) zygote is capable of giving rise to a viable offspring. By contrast, while haploid gametes conditions may give rise to embryos being parthenogenetic development of female-derived haploid cells (oocytes) these embryos typically stop developing before embryogenesis is completed. Such embryos may be produced spontaneously but more typically are produced by artificial activation of an oocyte. Such gynogenetic embryos are useful for the study of embryogenesis.
The production of properly haploid-derived pluripotent cell lines has previously been reported. For example, purported pluripotent haploid cells were allegedly created by obtaining eggs from 129 SvE or C57BL×CBA hybrid mice and activating them parthenogenetically following exposure to a 7% solution of ethanol in phosphate buffered saline (PBS). However upon examining the chromosomes of these early passage “Haploid” cell lines, all the cells were diploid with a modal number of 40 chromosomes (Kaufman et al., J. Embryol. Exp. Morphol. 73: 249-61 (1983)).
While it has been well reported that mammalian embryos may result from haploid genomes, such mammalian embryos have not been used for genetic analysis. Rather, to the best of the inventors' knowledge, prenatal genetic diagnosis is conventionally performed in utero or ex utero using apparent normal (diploid) embryos. However, in utero genetic diagnosis is invasive and can be dangerous to the developing fetus (e.g., amniocentesis and chorionic villi sampling). Fetuses diagnosed with disease can either be aborted or gestated to term, as in utero surgery and gene therapy are still highly risky and experimental.
In humans, ex utero genetic diagnosis is typically performed on embryos produced by in vitro fertilization (IVF) technologies. Typically one or two cells are taken from a recent embryo and tested for such diseases as cystic fibrosis (CF), sex-linked diseases, chromosomal abnormalities, fragile X syndrome, spinal muscular atrophy and myotonic dystrophy (de Die-Smulders et al., Ned. Tijdschr. Geneeskd. 142: 2441-4 (1998)). Preimplantation genetic diagnosis (PGD) can be performed using direct polymerase chain reaction (PCR) or nested PCR to diagnose the common ΔF508 mutation of CF (Cui et al., Mol. Hum. Reprod. 2: 63-1 (1996); and Ao et al., Prenat. Diagn. 16: 137-42 (1996)), as well as other diseases (Ben-Ezra, Clin. Lab. Med. 15: 95-815 (1995)). Genetic screening can also be done by single blastomere biopsy for rhesus (RhD) blood group typing of early cleavage stage embryos (Avner et al., Mol. Hum. Reprod. 2: 60-2 (1996)) or by blastocyst biopsy (Verlinsky et al., Bailieres Clin. Obstet. Gynaecol. 8: 177-96 (1994)). Primed in-situ labeling (PRINS) and in-situ hybridization can be used for detecting human chromosomal abnormalities for PGD (Pellestor et al., Mol. Hum. Reprod. 2: 135-8 (1996)). PGD has also been performed using fluorescence in situ hybridization (FISH) to prevent development of moles resulting from a fertilization of an inactive oocyte by a haploid X-bearing spermatozoon, which subsequently duplicates (Reubinoff et al., Hum. Reprod. 12: 805-8 (1997)). PGD can be performed on oocytes to diagnose single gene disorders by first polar body analysis and to identify oocytes that contain maternal unaffected genes (Verlinsky et al., Biochem. Mol. Med. 62: 182-7 (1997); Verlinsky et al., Curr. Opin. Obstet. Gynecol. 4: 720-5 (1992); and Verlinsky et al., Hum. Reprod. 5: 826-9 (1990)). In one case, individual spermatoza of a father with two affected infants with osteogenesis imperfecta, were separated by dilution and micromanipulation. A segment of the type I collagen gene containing the mutation was amplified using nested PCR and sequencing to detect the wild-type gene as well as genes with a single point mutation (Iida et al., Mol. Hum. Reprod. 2:131-4 (1996)). Methods of selecting sperm have been developed in response to use of intracytoplasmic sperm injection techniques (ICSI) (Meschede et al., Hum. Reprod. 10: 2880-6 (1995)). Sequential analysis of first and second polar body and multiplex PCR can lead accurate genetic diagnosis in comparison to the pitfalls encountered by single-cell DNA analysis (Richitsky et al., J. Assist. Reprod. Genet. 16: 192-8 (1999)).
Additional methods of genetic screening includes the detection or change in restriction fragment length polymorphisms (RFLPs), variable number of tandem repeat (VNTR) sequences and dinucleotide or other short tandem repeat (STR) sequences. Alternatively, allele specific amplification and allele specific ligation, utilizing primers complimentary to either the wild type or the mutant sequence, provide two alternative means for detection of specific mutations. Other methods are available to screen for the presence of mutations without identifying the specific mutation itself. These methods include single-strand conformational polymorphism (SSCP) analysis, denaturing gradient gel electrophoresis (DGGE), and mismatch cleavage analysis by enzymatic (RNAse A) or chemical (piperidine) means. See Fujimura, “Genetic Testing,” IN MOLECULAR BIOLOGY AND BIOTECHNOLOGY: A COMPREHENSIVE DESK REFERENCE 374-379 (Robert A. Meyers, ed., 1995).
Thus, based on the foregoing, it is evident that although research is ongoing in perfecting preimplantation genetic screening, as well as manipulation of embryos created in vitro, little progress has been achieved in the genetic screening of gametes or the genetic manipulation of gametes to be used to make transgenic animals.
Therefore, notwithstanding what has previously been reported in the literature, there exists a need for improved methods of genetic screening of gametes and genetically engineering haploid cells for preparing transgenic animals.